Recombinant Candida glabrata COPII coat assembly protein SEC16 (SEC16), partial

What is the functional role of SEC16 in Candida glabrata?

SEC16 serves as an essential scaffold protein for COPII vesicle formation at endoplasmic reticulum exit sites (ERES). Research has demonstrated that full-length SEC16 plays dual roles: it not only serves as a scaffold for COPII assembly but also performs a critical regulatory function in the late steps of COPII vesicle formation. The protein interacts with multiple COPII components and helps regulate the COPII-mediated ER-to-Golgi transport pathway, which is essential for secretory protein trafficking .

How does SEC16 interact with other COPII components?

SEC16 interacts with Sec23 and Sar1 through its C-terminal conserved region. These interactions are functionally significant as SEC16 hinders the ability of Sec31 to stimulate Sec23 GAP (GTPase-activating protein) activity toward Sar1. This regulatory mechanism ensures prolonged COPII coat association within preformed SEC16 clusters, which may lead to the formation of stable ERES. The interaction network between SEC16 and other COPII proteins establishes a mechanistic relationship between COPII coat assembly and ERES formation .

What structural features enable SEC16 to function in COPII assembly?

Purified SEC16 can self-assemble into homo-oligomeric complexes on planar lipid membranes, which is a crucial property for its function. This self-assembly capability allows SEC16 to create a platform for COPII component recruitment and assembly. The C-terminal conserved region is particularly important for protein-protein interactions with other COPII components. These structural features collectively contribute to SEC16's role in regulating Sar1 GTPase activity and maintaining the integrity of COPII-coated vesicles during their formation .

What are the optimal storage conditions for recombinant C. glabrata SEC16 protein?

The shelf life of recombinant SEC16 is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself. For optimal preservation:

• Liquid form: Store at -20°C/-80°C with a general shelf life of 6 months

• Lyophilized form: Store at -20°C/-80°C with an extended shelf life of 12 months

• Avoid repeated freezing and thawing

• For working stocks, store aliquots at 4°C for up to one week

For long-term storage, adding glycerol to a final concentration of 50% and aliquoting the protein is recommended before storing at -20°C/-80°C .

What reconstitution protocol is recommended for optimal SEC16 activity?

For reconstitution of recombinant SEC16:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) for long-term storage

• Aliquot to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C

The standard recommended final glycerol concentration is 50%, but researchers should optimize based on their specific experimental conditions .

What experimental approaches are most effective for studying SEC16 interactions with other COPII components?

To effectively study SEC16 interactions with COPII components:

• Protein-protein interaction assays: Co-immunoprecipitation, yeast two-hybrid assays, or pull-down assays can identify direct binding partners.

• Real-time PCR: For quantifying gene expression levels of SEC16 and interaction partners under different conditions. Studies have successfully used primers specific to the gene of interest (e.g., for CgDTR1, researchers used forward primer 5′-GGAGCCAAAATGAGAATGATATGTC−3′ and reverse primer 5′-ACCACCTTGAAATCGGTGATG−3′) .

• Fluorescence microscopy: Using GFP-tagged SEC16 expressed in either S. cerevisiae or C. glabrata cells allows visualization of the subcellular localization. Researchers have successfully used excitation and emission wavelengths of 395 and 509 nm respectively, with images captured using a cooled CCD camera .

• Membrane reconstitution assays: To observe SEC16 self-assembly into homo-oligomeric complexes on planar lipid membranes, which provides insights into how it creates ERES platforms .

How does SEC16 regulation of Sar1 GTPase activity impact vesicle dynamics?

SEC16 plays a sophisticated role in regulating Sar1 GTPase activity, which directly impacts COPII vesicle dynamics. Research has revealed that:

• SEC16 specifically hinders Sec31's ability to stimulate Sec23 GAP activity toward Sar1

• This inhibition prolongs the GTP-bound state of Sar1, which extends COPII coat association with the membrane

• The extended coat association allows for more complete vesicle formation and cargo capture

The mechanism creates a dynamic balance between coat assembly and disassembly. For researchers investigating this phenomenon, single-vesicle trafficking assays combined with real-time fluorescence microscopy can provide quantitative measurements of vesicle dynamics under various SEC16 concentrations or with SEC16 mutants lacking specific interaction domains .

How do strain-specific variations in C. glabrata affect SEC16 function and COPII vesicle formation?

Strain background significantly influences various aspects of C. glabrata biology, which may extend to SEC16 function. Research comparing strains like CBS138 and BG2 has revealed:

• Metabolic differences between strains that play biologically meaningful roles during infection

• Strain-dependent genetic rewiring affecting cell wall architecture and biogenesis

• Variations in virulence gene expression and function depending on strain background

These findings suggest that researchers must carefully consider strain background when studying SEC16. Comparative genomic analyses between different C. glabrata strains could reveal variations in SEC16 sequence, expression, or interaction networks. When designing experiments, researchers should characterize SEC16 function across multiple reference strains to ensure robust findings .

What methodological approaches can resolve contradictory data on SEC16 function between in vitro and in vivo systems?

Researchers often encounter discrepancies between in vitro biochemical data and in vivo observations regarding SEC16 function. To reconcile such contradictions:

• Correlated light and electron microscopy (CLEM): This technique allows visualization of SEC16-mediated ERES formation in both fixed and living cells, providing structural details that can be correlated with functional data.

• Reconstituted systems of increasing complexity: Starting with purified components, then moving to synthetic membranes, permeabilized cells, and finally intact cells to identify stage-specific behaviors of SEC16.

• Quantitative proteomics: Comparing SEC16 interaction partners between in vitro and in vivo conditions can reveal context-dependent regulatory factors.

• Gene editing technologies: Creating specific mutations or truncations in SEC16 to systematically test which domains are responsible for discrepancies between in vitro and in vivo systems .

How can researchers distinguish between essential housekeeping functions of SEC16 and potential pathogenesis-specific roles?

This distinction requires sophisticated experimental design:

• Conditional expression systems: Using copper-inducible promoters (like the MTI promoter) to create titratable SEC16 expression, allowing researchers to identify the threshold between essential function and pathogenesis-specific effects

• Domain-specific mutations: Creating targeted mutations in specific SEC16 domains to separate housekeeping functions from specialized roles in pathogenesis

• Comparative transcriptomics: Analyzing SEC16 expression patterns during commensal versus invasive growth, potentially using hemolymph recovery methods similar to those used for other C. glabrata virulence factors

• Heterologous expression: Expressing C. glabrata SEC16 in S. cerevisiae to identify functions unique to the pathogenic species versus shared housekeeping roles

How does genetic diversity within the C. glabrata population impact SEC16 function and potential as a therapeutic target?

The significant genetic diversity in C. glabrata populations has important implications for SEC16 research:

• Comparative genomic analyses have revealed marked genetic diversity between clinical isolates, including variations in virulence genes and drug targets

• Microevolution during prolonged or recurrent infection could potentially affect SEC16 sequence or regulation

• Mitochondrial genome diversity, which is particularly pronounced in C. glabrata, may indirectly impact SEC16 function through altered cellular energetics

When evaluating SEC16 as a potential therapeutic target, researchers must:

• Sequence SEC16 across diverse clinical isolates to identify conserved regions suitable for targeting

• Test potential interventions against multiple strain backgrounds to ensure broad efficacy

• Consider the potential for adaptive mutations in response to SEC16-targeted therapies

What quality control parameters should be monitored when working with recombinant C. glabrata SEC16?

Researchers should monitor several key parameters:

The standard for commercially available recombinant SEC16 is >85% purity as assessed by SDS-PAGE. Researchers should verify this using their own quality control protocols before experimentation .

What experimental controls are essential when studying SEC16 in COPII vesicle formation assays?

Several critical controls should be included in any SEC16-focused COPII vesicle formation study:

• Positive controls: Include well-characterized COPII components (Sar1, Sec23/24, Sec13/31) in assays to verify system functionality

• Negative controls:

  • Omission of GTP to confirm GTP-dependency

  • Heat-inactivated SEC16 to confirm specific activity

  • Irrelevant proteins of similar size/charge to confirm specificity

• Concentration gradients: Testing multiple concentrations of SEC16 to identify dose-dependent effects

• Domain mutants: SEC16 constructs with mutations in key interaction domains to validate mechanism-specific findings

How can researchers overcome challenges in expressing and purifying full-length C. glabrata SEC16?

Full-length SEC16 expression and purification presents several challenges that researchers can address through these methodological approaches:

• Expression systems optimization:

  • E. coli systems often yield partial SEC16 preparations with good purity (>85%)

  • For full-length protein, consider baculovirus-insect cell expression systems which better handle large eukaryotic proteins

  • Codon optimization for the expression host improves yield

• Solubility enhancement:

  • Fusion tags like MBP (maltose-binding protein) or SUMO can improve solubility

  • Expression at lower temperatures (16-18°C) reduces inclusion body formation

  • Co-expression with chaperones may improve folding

• Purification strategy:

  • Multi-step purification combining affinity, ion exchange, and size exclusion chromatography

  • Including stabilizing agents such as glycerol (5-50%) in all buffers

  • Avoiding repeated freeze-thaw cycles by preparing single-use aliquots

How do post-translational modifications regulate SEC16 function in C. glabrata?

While specific data on C. glabrata SEC16 post-translational modifications (PTMs) is limited, research in related organisms suggests several important regulatory mechanisms:

• Phosphorylation likely regulates SEC16 activity in response to cellular conditions and stress

• PTMs may control SEC16 oligomerization and interaction with COPII components

• Stress-responsive modifications could link secretory pathway function to pathogenesis

Research methods to investigate these PTMs include:

• Mass spectrometry-based phosphoproteomics under various stress conditions

• Site-directed mutagenesis of putative modification sites

• Comparative analysis of PTM patterns between commensal and pathogenic growth states

What imaging techniques are most suitable for visualizing SEC16 dynamics during COPII vesicle formation in living cells?

Advanced imaging techniques for studying SEC16 dynamics include:

• Super-resolution microscopy: Techniques like PALM, STORM, or SIM can resolve SEC16 clusters below the diffraction limit, revealing nanoscale organization

• Lattice light-sheet microscopy: Enables long-term 3D imaging with minimal phototoxicity, ideal for capturing the entire lifecycle of ERES formation

• Fluorescence recovery after photobleaching (FRAP): Quantifies SEC16 dynamics and turnover at ERES

• Single-molecule tracking: Reveals the behavior of individual SEC16 molecules during ERES assembly

For C. glabrata specifically, researchers have successfully used fluorescence microscopy with SEC16-GFP fusion proteins expressed under copper-inducible promoters, with excitation and emission wavelengths of 395 and 509 nm respectively .

How might understanding SEC16 function contribute to novel antifungal development strategies?

SEC16's essential role in secretory pathway function presents potential opportunities for antifungal development:

• Selective inhibition: Identifying structural or functional differences between fungal and human SEC16 homologs could enable selective targeting

• Combination therapy: SEC16 inhibitors might sensitize C. glabrata to existing antifungals by disrupting cell wall maintenance

• Virulence attenuation: Partial inhibition of SEC16 function could reduce virulence without strong selective pressure for resistance

Experimental approaches should include:

• Comparative structural analysis between fungal and human SEC16

• High-throughput screening for compounds that disrupt SEC16-COPII interactions

• In vivo testing using infection models like G. mellonella larvae to assess efficacy and specificity